Immune evasion by HIV-1 accessory proteins likely contributes to the failure of host immunity to control the infection and may complicate approaches to eradication. We will test the hypothesis that inhibiting viral evasion of host immunity can contribute to a curative treatment strategy. Recent data suggest that reactivation of viral gene expression is not sufficient to cause the death of cells latently infected with HIV-1 cell-death requires the killing-activity of cytotoxic T lymphocytes (CTL). Consequently, our strategy focuses on HIV-1 Nef, which provides viral evasion of CTL-activity. Nef prevents class I MHC from reaching the plasma membrane, decreasing the concentration of viral antigens at the cell surface and inhibiting the killing of infected cells by CTL. Interference with this activity o Nef should empower CTL and facilitate the eradication of infected cells from the host. In principle, this strategy applies to reservoir cells expressing low levels of viral antigens as well as to CD-positive memory T cells in which latent virus is reactivated pharmacologically. During the R21 phase of this proposal, we will validate the importance of Nef in viral resistance to eradication by showing that primary T cells in which virus is reactivated from latency display reduced surface levels of MHC-I and that this reduction is Nef-dependent and associated with resistance to CTL-mediated killing. Concurrently, we will use our recent knowledge of how Nef modulates MHC-I at the molecular and structural levels to identify small molecules capable of inhibiting this activity. We will exploit our X-ray crystallographic model of the complex formed by Nef, the cytoplasmic domain of the MHC-I ?-chain, and the ? subunit of the endosomal clathrin adaptor AP1 to design prototypic peptide inhibitors and to devise a high throughput screen capable of identifying small molecule lead compounds. During the R33 phase, we will broaden our approach to drug discovery by including a novel computational method that matches semi-rigid scaffold molecules displaying amino acid R groups to the structure of the complex. We will evaluate leads from this approach as well as from our high throughput screen using cell- based secondary screens and optimize them structurally. Lastly, we will use these small molecules in in vitro, ex vivo, and in vivo settings o establish that inhibiting Nef-mediated immune evasion can facilitate viral eradication.